Fermented milk for skin improvement and/or treatment and process for producing the same

ABSTRACT

The present invention provides a composition and fermented milk, which is derived from natural product, is highly safe, exhibits improvement effect and/or therapeutic effect on skin, and comprises fermented lactic acid bacteria excellent in manufacturability. Disclosed are: use of  Streptococcus thermophilus  OLS3059 in skin improvement and/or treatment; a composition for skin improvement and/or treatment comprising  Streptococcus thermophilus  OLS3059 and/or a culture thereof; food and drink comprising the composition; fermented milk for skin improvement and/or treatment prepared by using  Lactobacillus delbrueckii  subspecies  bulgaricus  and  Streptococcus thermophilus  OLS3059; and the fermented milk for skin improvement and/or treatment further comprising collagen peptide and/or ceramide.

TECHNICAL FIELD

The present invention relates to a composition having high improvementeffect and/or therapeutic effect on skin, food and drink and fermentedmilk using the same and particular to a composition and fermented milkcomprising lactic acid bacteria excellent in manufacturability.

BACKGROUND ART

As compositions and food and drink for the purpose of skin improvement,treatment etc., there are those containing lactic acid bacteria orcultures thereof.

Related arts directed to such compositions and food and drink aredescribed below:

an atopic dermatitis suppressant and food using specific lactic acidbacteria (Patent Document 1);

a humectant having an improvement effect on skin, which uses a cultureof specific lactic acid bacteria and a cosmetic composition effective atprevention and treatment of rough skin and dry skin and giving “faircomplexion” and “supple state” to skin (Patent Document 2);

use of specific lactic acid bacteria and cultures thereof inwell-balanced immune system of skin as well as food or the likecomprising lactic acid bacteria and a culture thereof (Patent Document3);

weight-reducing food expected to achieve improvement in laxation,recovery from fatigue, alleviation of stiff neck, etc. in addition toimprovement in skin by using lactic acid bacteria or a culture thereofin combination collagen peptide or ceramide (Patent Document 4).

As described above, it was found that lactic acid bacteria and culturesthereof were found to have an improvement effect and therapeutic effecton skin, and actually, industrial application thereof has conventionallybeen expected. Some specific studies have been made, and it is estimatedthat there are actually some commercial products thereof.

However, examination of the relationship between lactic acid bacteriaand skin or the relationship between a composition comprising, forexample, collagen peptide and ceramide added to lactic acid bacteria andskin is insufficient in conventional studies. Accordingly, there is ashortage of scientific grounds for the improvement effect of lactic acidbacteria or fermented milk on skin etc.

Conventional lactic acid bacteria having an improvement effect on skinetc., when producing fermented milk, do not form curd, or the time forforming curd is significantly long. In this case, lactic acid bacteriahaving an improvement effect on skin but poor in manufacturability offermented milk and lactic acid bacteria excellent in manufacturabilityfor forming curd are separately managed and the fermentation time isprolonged, and thus the manufacturing time is increased as a whole.Conventional lactic acid bacteria having an improvement effect on skinetc. are not viable bacteria but killed bacteria or highly thermostablelimited bacteria. That is, conventional lactic acid bacteria having animprovement effect on skin etc. are not suitable for large-scaleproduction (commercial production) of fermented milk etc.

Roughly speaking, manufacturing processes shown below will be necessarywhen conventional lactic acid bacteria regarded as having an improvementeffect on skin etc. are used to produce fermented milk. That is, (1)lactic acid bacteria having an improvement effect on skin etc. arecultured alone. (2) The lactic acid bacteria (cultured microbial bodies)are concentrated to a predetermined density by centrifugation orseparation through a membrane, or their culture is sterilized by heatingor dried in order to improve the storage stability of the lactic acidbacteria, depending on the situation. (3) Other lactic acid bacteriaforming curd, their concentrated cultured microbial bodies, or thosesterilized by heating or dried are added to blended milk beforefermentation and subjected to mixed culture.

These manufacturing processes are complicated to regulate properly, andthe manufacturing costs and the manufacturing time are increased, andthere is a high probability of contamination. Fermented milk suitingconsumer's liking and excellent in taste is hardly obtainable.

Patent Document 1: Japanese Patent Application Laid-Open No. 2000-095697Patent Document 2: Japanese Patent Application Laid-Open No. 2003-081808Patent Document 3: Japanese Patent Application Laid-Open No. 2004-510740Patent Document 4: Japanese Patent Application Laid-Open No. 2004-254632DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

The present invention was made in view of the related art and problemsdescribed above, and the object of the present invention is to provide acomposition which is derived from natural products, is highly safe andhas high improvement effect and/or therapeutic effect on skin, acomposition suitable for use in skin improvement and/or treatment, foodand drink and fermented milk having high improvement effect and/ortherapeutic effect on skin, comprising the composition, particularly acomposition and fermented milk comprising lactic acid bacteria excellentin manufacturability.

Means for Solving the Problem

As a result of extensive study in view of the problem described above,the present inventors conducted an in vitro test for the purpose ofselecting lactic acid bacteria having high skin improvement effect andtreatment effect from those used in preparation of fermented milk, andthereby found that Streptococcus thermophilus OLS3059 has particularlyhigh skin improvement and/or treatment effect, and the present inventionwas arrived at.

That is, the invention according to claim 1 is use of Streptococcusthermophilus OLS3059 in skin improvement and/or treatment.

The invention according to claim 2 is a composition for skin improvementand/or treatment, which comprises Streptococcus thermophilus OLS3059and/or a culture thereof.

The invention according to claim 3 is the composition for skinimprovement and/or treatment according to claim 2, which furthercomprises Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1and/or a culture thereof.

The invention according to claim 4 is food and drink comprising thecomposition of claim 2.

The invention according to claim 5 is fermented milk for skinimprovement and/or treatment prepared by using Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059.

The invention according to claim 6 is the fermented milk for skinimprovement and/or treatment according to claim 5, which furthercomprises collagen peptide and/or ceramide.

The invention according to claim 7 is a process for producing thefermented milk for skin improvement and/or treatment of claim 5 or 6,which comprises preparing raw milk, adding Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059 as startersto the milk, charging the resulting milk into a container, andfermenting it to form curd followed by cold-storage thereof.

The invention according to claim 8 is a process for producing thefermented milk for skin improvement and/or treatment of claim 5 or 6,which comprises preparing raw milk, adding Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059 as startersto the milk, fermenting the resulting milk to form curd, disrupting thecurd, and charging it into a container followed by cold-storage thereof.

The invention according to claim 9 is a process for producing thefermented milk for skin improvement and/or treatment of claim 5 or 6,which comprises preparing raw milk, adding Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059 as startersto the milk, fermenting the milk to form curd, disrupting the curd,cooling it, and charging it into a container followed by cold-storagethereof.

In the present invention, the phrase “skin improvement effect and/ortreatment effect” refers to an improvement effect on at least one stateselected from skin gloss, firmness, dullness, fleck, sagging,perceptible pores of the skin, fine texture, wrinkle, degree of dryness,stickiness, transparency, redness, spreading of foundation cream on theskin, swelling, perceptible dark circles under eyes, eruption, moistureretention, and deposition of pigment.

The phrase “skin improvement and/or treatment” refers to improvement ofat least one state selected from skin gloss, firmness, dullness, fleck,sagging, perceptible pores of the skin, fine texture, wrinkle, degree ofdryness, stickiness, transparency, redness, spreading of foundationcream on the skin, swelling, perceptible dark circles under eyes,eruption, moisture retention, and deposition of pigment.

The phrase “skin improvement and/or treatment” includes amelioration ofskin conditions caused by atopic dermatitis and various kinds ofallergic dermatitis, as well as suppression of development of suchdermatitis.

The phrase “for skin improvement and/or treatment” is intended for theproduct of the invention to be used for the purpose of exhibiting theskin improvement effect and/or treatment effect, and is particularlyintended for it to be suitable for use in exhibiting the skinimprovement effect and/or treatment effect.

In the present invention, Lactobacillus delbrueckii subspeciesbulgaricus is used in combination with Streptococcus thermophilusOLS3059 and used as the starter in production of fermented milk, therebyallowing the product to contain all lactic acid bacteria having anability to form curds, and for example, there are lactic acid bacteriaobtained by isolation from plain yoghurt, hard yoghurt and soft yoghurtmanufactured by Meiji Dairies Corporation.

In the present invention, Streptococcus thermophilus OLS3059 has beendeposited under Accession No. FERM P-15487 (indication foridentification: Streptococcus thermophilus OLS3059) since Feb. 29, 1996(accession date), with International Patent Organism Depositary (IPOD),National Institute of Advanced Industrial Science and Technology (AIST),Japan.

Transfer of this original deposition to deposition based on the Budapesttreaty was conducted on Nov. 29, 2006, under Accession No. FERM BP-10740with the International Depository Authority.

The bacterial state of this Streptococcus thermophilus OLS3059 is asfollows:

Colony form: cylindrical, trapezoidal and smooth.

(BL plate (horse blood-free), culture temperature: 37° C., culture time:48 h)

Bacterial form: coccoid

Gram staining: positive

Oxygen requirement: aerobic

Sugar assimilability

Ribose −

Mannitol −

Sorbitol −

Lactose +

Trehalose −

Raffinose −

Maltose −

Melibiose −

Melezitose −

Arabinose −

In the present invention, Lactobacillus delbrueckii subspeciesbulgaricus OLL1073R-1 has been deposited under Accession No. FERMP-17227 (indication for identification: Lactobacillus delbrueckiisubspecies bulgaricus OLL1073R-1) since Feb. 29, 1999 (accession date),with International Patent Organism Depositary (IPOD), National Instituteof Advanced Industrial Science and Technology (AIST), Japan.

Transfer of this original deposition to deposition based on the Budapesttreaty was conducted on Nov. 29, 2006, under Accession No. FERM BP-10741with the International Depository Authority.

The bacterial state of Lactobacillus delbrueckii subspecies bulgaricusOLL1073R-1 is as follows:

Colony form: opaque and rough.

(BL agar plate, culture temperature: 37° C., culture time: 48 h)

Bacterial form: rod-shaped

Gram staining: positive

Oxygen requirement: facultative anaerobic

Sugar assimilability

Ribose −

Mannitol −

Sorbitol −

Lactose +

Trehalose −

Raffinose −

Maltose −

Melibiose −

Melezitose −

Arabinose −

EFFECT OF THE INVENTION

The present invention can provide use of Streptococcus thermophilusOLS3059 in skin improvement and/or treatment.

According to the present invention, there can be provided a compositionwhich is derived from natural products, is highly safe and has high skinimprovement effect and/or treatment effect, a composition suitable foruse in skin improvement and/or treatment, food and drink and fermentedmilk having a skin high improvement effect and/or treatment effect,comprising the composition, particularly a composition and fermentedmilk comprising lactic acid bacteria excellent in manufacturability.

According to the present invention, there can be provided fermented milkhaving a high skin improvement effect and treatment effect on humans forwhom it exhibited a high intestinal function (bowel movement)improvement effect and/or treatment effect, as well as a process forproducing the same.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides use of Streptococcus thermophilus OLS3059in skin improvement and/or treatment. That is, the present inventorsmade extensive study, and as a result, they found that thatStreptococcus thermophilus OLS3059 has a particularly high skinimprovement effect and/or treatment effect in an in vitro test conductedfor the purpose of selecting lactic acid bacteria having a high skinimprovement effect and treatment effect from those used in preparationof fermented milk, and the present invention was thereby completed.

Then, the composition of the present invention is a composition for skinimprovement and/or treatment, which comprises Streptococcus thermophilusOLS3059 and/or a culture thereof. The composition for skin improvementand/or treatment according to the present invention is derived fromnatural products, and is thus highly safe and suitable for theseapplications.

The present inventors, then conducted an in vitro test for the purposeof selecting lactic acid bacteria having a high skin improvement effectand/or treatment effect from those having an ability to form curds andbeing excellent in productivity and usable in preparation of fermentedmilk, found that Streptococcus thermophilus OLS3059 has a particularlyhigh skin improvement effect and/or treatment effect as described above.

The composition for skin improvement and/or treatment comprisingStreptococcus thermophilus OLS3059 and/or a culture thereof according tothe present invention can be a composition further comprisingLactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 and/or aculture thereof.

In the inventors' experiments, it was recognized that Streptococcusthermophilus OLS3059 and/or a culture thereof, when used in combinationwith Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1extracellularly producing viscous polysaccharides, can also have a highskin improvement effect and/or treatment effect and is suitable forthese applications.

Streptococcus thermophilus OLS3059 is used in, for example, Bulgarianyoghurt LB81 (registered trademark) manufactured by Meiji DairiesCorporation.

Streptococcus thermophilus OLS3059 and/or a culture thereof,Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 and/or aculture thereof, include bacteria themselves obtained by isolation andpurification of the culture, the culture containing the bacteria, andany materials obtained by treating the culture, including an extract ofthe bacteria, a supernatant of the culture, a concentrate thereof, aconcentrated or dried material thereof, and if necessary, a dilutedliquid or material thereof, etc.

The above-mentioned thermophilus OLS3059 whether viable bacteria orkilled bacteria has a high skin improvement effect and/or treatmenteffect and can be used in skin improvement and/or treatment. Theabove-mentioned bulgaricus OLL1073R-1 can also be used regardless ofwhether it is viable bacteria or killed bacteria. For thermophilusOLS3059 and bulgaricus OLL1073R-1, the culture method, extractionmethod, separation method, concentration method, drying method, dilutionmethod etc. are not particularly limited. The medium for culturing thebacteria usually contains skim milk, whey, milk protein such as casein,sugars, yeast extract etc., and as the culture method, a wide variety ofgeneral aerobic or anaerobic methods can be used depending on the case.It is also possible to use a neutralization culture method wherein theculture temperature is set for example at 35 to 45° C., and the mediumpH is kept in the neutral to acidic range, for example in the range ofabout pH 5 to 6 by using an alkali such as sodium hydroxide duringculture. Besides the neutralization culture method, an arbitrary culturemethod such as a batch culture method can be used, and after culture, aculture or its supernatant can be subjected if necessary toconcentration, drying, dilution etc. The culture may be separated bycentrifugation or through a membrane into a supernatant and thebacteria, and the bacteria can also be recovered in a concentratedstate. Then, the bacteria are subjected to sonication or enzymetreatment, whereby bacterial components can be extracted, or theculture, its supernatant, the bacteria, their extract etc. can be dried.These can be used as the active ingredient in the composition of thepresent invention.

The composition of the present invention can be compounded with anarbitrary component and used as an external preparation in a usualmanner. For example, the composition can be blended if necessary withvarious components used in usual cosmetics, medicated cosmetics andpharmaceutical preparations and with an oil solution, a thickener, apreservative, an emulsifying agent, a pigment, a pH adjusting agent, anantioxidant and a perfume and used arbitrarily as cream, an emulsion,cosmetics, a foundation, a lotion and a shampoo depending on the object.

The food and drink of the present invention contain the composition ofthe present invention. The drink and food of the present invention has ahigh skin improvement effect and/or treatment effect and is suitable forthese applications.

Because the active ingredient in the composition of the presentinvention is lactic acid bacteria which have been actually used in food,the composition is problematic with respect to safety and can beadministered orally or parenterally as food, drink and a pharmaceuticalpreparation.

In this case, Streptococcus thermophilus OLS3059 can be used alone or asa mixture with usual human-diet components. Each of the thermophilusOLS3059 or bulgaricus OLL1073R-1 can be used alone, or these may becombined and used as a mixture with usual human-diet components.Alternatively, the thermophilus OLS3059, or a combination ofthermophilus OLS3059 and bulgaricus OLL1073R-1, can be formed into apharmaceutical preparation by mixing it with a wide variety of usualauxiliary substances used in production of an oral pharmaceuticalpreparation and/or a parenteral pharmaceutical preparation.

When Streptococcus thermophilus OLS3059, or a combination ofthermophilus OLS3059 and bulgaricus OLL1073R-1, is used as a mixturewith usual human-diet components or with a wide variety of usualauxiliary substances used in production of an oral pharmaceuticalpreparation and/or a parenteral pharmaceutical preparation, the(blending) amount of thermophilus OLS3059 can be determined suitably inconsideration of the administration method and the age and weight of aperson who ingests the food and drink of the invention, and is at least10⁹ cfu/day, preferably at least 10¹⁰ cfu/day.

The food and drink of the present invention include those containing thecomposition of the present invention described above, which include notonly drink and tablets but also biscuits, bread, sponge cakes, fermentedmilk, yoghurt, cheese, butter, margarine, cream and jelly. With respectto the form and method in which the food and drink of the presentinvention are used, there are the form and method in which thecomposition of the present invention is added directly to the food anddrink described above. In an alternative form and method, the yoghurt,cheese, butter etc. that are the food and drink of the invention thusobtained are further added to food and drink.

For the purpose of exhibiting the skin improvement effect and/ortreatment effect, the food and drink of the present invention can beused as usual food and drink but also as food for specified health use,nutrient functional food, health food etc.

The drink and food of the present invention have a skin improvementeffect and/or treatment effect and can thus be prepared as food anddrink provided with an indication to the effect that it is used for skinimprovement and/or treatment. The method of indication includesdescription on a pamphlet, advertisement, etc., in addition toindication by a package for the food and drink.

The composition or the food and drink according to the presentinvention, when used as a pharmaceutical preparation, can be used in theform of tablets, capsules, granules, powder or syrup. In this case,composition or the food and drink can be formed into a pharmaceuticalpreparation by using usual auxiliary substances in addition to anexcipient (that is, a base), a binder, a disintegrating agent, alubricant, a flavoring substance, a smell corrective, a solubilizingagent, a suspending agent, and a coating agent.

The fermented milk of the present invention is fermented milk for skinimprovement and/or treatment, which is prepared by using Lactobacillusdelbrueckii subspecies bulgaricus and Streptococcus thermophilusOLS3059.

The fermented milk for skin improvement and/or treatment according tothe present invention has a high skin improvement effect and/ortreatment effect because it is prepared by using Streptococcusthermophilus OLS3059 having a high skin improvement effect and/ortreatment effect from lactic acid bacteria having an ability to formcurds, being excellent in productivity and usable in preparation offermented milk. The fermented milk of the present invention has a highskin improvement effect and/or treatment effect and is suitable forthese applications.

The above-mentioned thermophilus OLS3059 is combined with the bulgaricusbacteria to regulate lactic acid bacteria; for example, these arecombined and used as the starter, whereby curds can be formed in arelatively short time.

For example, lactic acid bacteria obtained by isolation from plainyoghurt, hard yoghurt and soft yoghurt manufactured by Meiji DairiesCorporation can be used as Lactobacillus delbrueckii subspeciesbulgaricus in the fermented milk for skin improvement and/or treatmentaccording to the present invention. The fermented milk of the presentinvention prepared by using a combination of these bulgaricus bacteriaand thermophilus bacteria is characterized by being excellent in taste,having good flavor and smooth feeling on the tongue, and forming strongcurds to be hardly broken during transportation of the product.

The fermented milk for skin improvement and/or treatment according tothe present invention, prepared by using the above-mentionedLactobacillus delbrueckii subspecies bulgaricus and Streptococcusthermophilus OLS3059, can further contain collagen peptide and/orceramide.

When the collagen peptide- and ceramide-containing fermented milk of theinvention is compared with the collagen peptide- and ceramide-freefermented milk of the invention, as will be described later, thecollagen peptide- and ceramide-containing fermented milk tended to havea higher skin improvement effect and/or treatment effect. That is, theskin improvement effect and/or treatment effect attained by thefermented milk of the invention prepared by using the bulgaricusbacteria and thermophilus OLS3059 only was lower than that attained bythe fermented milk of the invention prepared by using the bulgaricusbacteria and thermophilus OLS3059 having collagen peptide and/orceramide further mixed therewith. This higher effect is consideredattributable to the synergistic effect of mixing collagen peptide and/orceramide with the lactic acid bacteria having a high skin improvementeffect and/or treatment effect (Streptococcus thermophilus OLS3059).

From the viewpoint of exhibiting such synergistic effect, the collagenpeptide contained in the fermented milk is preferably 500 mg % or more,more preferably 700 mg % or more, still more preferably 900 mg % ormore. From the viewpoint of exhibiting the effect, the ceramidecontained in the fermented milk is preferably 100 μg % or more, morepreferably 200 μg % or more, still more preferably 250 μg % or more.

From the viewpoint of the synergistic effect for skin improvement effectand/or treatment effect, it is preferable to add, to the fermented milkof the present invention, not only collagen peptide and/or ceramide butalso various active ingredients used in foods, drinks and pharmaceuticalpreparations for metabolic improvement, such as sphingomyelin,isoflavone, chondroitin and hyaluronic acid.

To find a factor influencing the skin improvement effect and/ortreatment effect of the fermented milk for skin improvement and/ortreatment according to the present invention, the inventors made furtherdetailed examination in a human test, and as a result, they found thatthe fermented milk of the invention described above has a higher skinimprovement effect and/or treatment effect on humans for whom it showsan improvement effect on regulation of intestinal functions, which isregarded as an action typical of fermented milk.

Specifically, the collagen- and ceramide-containing fermented milk forskin improvement and/or treatment according to the present invention andthe collagen- and ceramide-free fermented milk for skin improvementeffect and/or treatment according to the present invention were used andcompared with each other in respect of the improvement effect onregulation of intestinal functions and the improvement effect on theskin, thereby attaining the finding described above.

That is, the fermented milk for skin improvement and/or treatmentaccording to the present invention is characterized in that it exhibitsa higher skin improvement effect and/or treatment effect on humans forwhom it exhibits a high intestinal function regulation (bowel movement)improvement effect and/or treatment effect.

The process for producing fermented milk according to the presentinvention wherein the fermented milk for skin improvement and/ortreatment according to the present invention is produced, comprises:

preparing raw milk, adding Lactobacillus delbrueckii subspeciesbulgaricus and Streptococcus thermophilus OLS3059 as starters to themilk, charging the resulting milk into a container, and fermenting it toform curd followed by cold-storage thereof.

Alternatively, the process comprises preparing raw milk, addingLactobacillus delbrueckii subspecies bulgaricus and Streptococcusthermophilus OLS3059 as starters to the milk, fermenting the resultingmilk to form curd, disrupting the curd, and charging it into a containerfollowed by cold-storage thereof;

or the process comprises preparing raw milk, adding Lactobacillusdelbrueckii subspecies bulgaricus and Streptococcus thermophilus OLS3059as starters to the milk, fermenting the milk to form curd, disruptingthe curd, cooling it, and charging it into a container followed bycold-storage thereof.

In preparation of raw milk before addition of Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059 as thestarter to the milk, the raw milk is prepared and subjected totreatments such as homogenization, sterilization and cooling in the samemanner as in a conventional process of producing fermented milk.

In the process for producing the fermented milk of the presentinvention, Streptococcus thermophilus OLS3059 having a high skinimprovement effect and/or treatment effect, selected from lactic acidbacteria having an ability to form curds, being excellent inproductivity and being used in production of fermented milk, is combinedwith the bulgaricus bacteria and used as the starter.

Accordingly, the problem of the conventional lactic acid bacteria havinga skin improvement effect etc. has been solved. Conventionally, lacticacid bacteria having an improvement effect on skin etc. but poor inmanufacturability of fermented milk, and lactic acid bacteria excellentin manufacturability for forming curds, are separately managed and thefermentation time is prolonged, so there is a problem that themanufacturing time is increased as a whole. That is, conventional lacticacid bacteria having an improvement effect on skin etc. are not suitablefor large-scale production (commercial production) of fermented milketc.

However, the process for producing the fermented milk according to thepresent invention has solved the problem described above and can thus beapplied to commercial production. It is important for commercialproduction that the process is simple with a fewer number of steps.

In the process for producing the fermented milk of the presentinvention, homogenization in the process for producing raw milk can becarried out before and/or after sterilization.

As described above, cooling after fermentation may be carried out afterformation of curd or disruption of curd, or after filling in acontainer.

Disruption of curd can be followed by adding or mixing fruit flesh,vegetables, various sauces and/or various additives.

In the process for producing the fermented milk of the presentinvention, the fermentation time is 8 hours or less, preferably 6 or 5hours or less, more preferably 4 hours or less, in such a range as to beusable in usual production of fermented milk.

The fermented milk for skin improvement and/or treatment according tothe present invention can be regularly used and easily and successivelyingested owing to its extremely excellent flavor. The skin improvementeffect and treatment effect can thereby be further increased.

Hereinafter, the present invention is described by reference topreferable examples, but the present invention is not limited thereto.

Example 1 Verification of Inhibitory Effect on Atopic Dermatitis in anIn Vitvo Test

Four-week-old female NC/Nga mice (manufactured by Nippon SLC Co., Ltd.)were acclimated and raised and then divided into the following 4 groups:

a distilled water administration group (n=10),

a Streptococcus thermophilus OLS3059 (also referred to hereinafter asOLS3059; isolated from Bulgarian yoghurt LB81 (registered trademark)manufactured by Meiji Dairies Corporation) administration group (n=10),a dermatitis non-induction group (n=7), and a Lactobacillus rhamnosusATCC 53103 (also referred to hereinafter as ATCC 53103) administrationgroup (n=10) as a control for comparison with the OLS3059 administrationgroup.

Feed (trade name: CRF-1, manufactured by Oriental Yeast Co., Ltd.) anddistilled water were freely given to the mice.

ATCC 53103 is a lactic acid bacterium known to be effective againstatopic dermatitis.

During the test period, distilled water or each lactic acid bacteriumwas orally administered with a probe to the 3 groups excluding thedermatitis non-induction group. According to a method described later,induction of dermatitis was conducted every day from Day 0 (that is, 7days after the test was initiated). That is, for the test period (fromthe 1st day (=Day −7) to the 21st day (=Day 14)), distilled water wasadministered orally to the stomach of the distilled water administrationgroup every day (1 mg/day/mouse) and an aqueous suspension of each kindof lactic acid bacterium was administered in the same manner to thestomach of the lactic acid bacterium administration group (1mg/day/mouse). The suspension of each kind of lactic acid bacterium wasprepared by suspending a lyophilized product of the bacterium subjectedto heat treatment (temperature 75° C. for 60 minutes). In the suspensionof each kind of lactic acid bacterium, the density of bacteria was7.13×10⁸ cfu/g for ATCC 53103 or 8.56×10⁷ cfu/g for OLS3059.

Dermatitis was induced from 7 days (Day 0) after the test was initiated,and a change in development was scored. Serum was collected 15 days (Day15) after the dermatitis was induced, and the IgE level was measured bythe ELISA method. The IgE level was measured by using an antibody(manufactured by Pharmingen) in accordance with a modification toPharmingen' recommended protocol.

A disrupted mite liquid was prepared in the following manner. That is,the whole of a mite Dermatophagoides pteronyssinus (Mite-Dp,manufactured by LSL) was defatted with anhydrous ether, then distilledwater was added thereto, and the mite was disrupted by sonication. Then,the resulting sample was centrifuged to give a water-soluble fractionwhich was then lyophilized. This lyophilized sample was dissolved indistilled water to prepare a solution with a protein concentration of4.5 mg/ml (in terms of BSA, determined by DC protein assay fromBIO-RAD).

The dermatitis induction test was carried out using a modification tothe method of Kaino et al. (Tetsushi Kaino et al., Allergy 50:1152-1162,2001). A dermatitis induction site (head, auricle and cervical region)was dehaired with a hair clipper, and an aqueous solution of SDS (4%)was applied 20 times with a brush onto the dermatitis induction site ofevery mouse. The amount of the SDS solution thus applied 20 times wasabout 60 μl in total. After the aqueous solution of SDS was dried, thedisrupted mite liquid and distilled water were applied 20 times in thesame manner to the dermatitis induction group and the dermatitisnon-induction group, respectively. The above-described operation wasrepeated every day as one set thereby inducing dermatitis and repeatedlycarried out on consecutive days from 7 days (Day 0) after the test wasinitiated to the day when the test was terminated.

Measurement of IgE was carried out in the following method. That is, ananti-IgE antibody for primary antibody (manufactured by Pharmingen) wasadded at 2 μg/ml to a 96-well plate and left for 1 hour at a temperatureof 37° C. After washing with Tween 20/PBS (PBS-Tween, 0.05%), BSA/PBS(1%) was added and left at room temperature for 30 minutes. Afterwashing with PBS-Tween, a serum sample and IgE for preparation ofstandard curve (manufactured by Pharmingen) were added and left at roomtemperature for 30 minutes. After washing with PBS-Tween,biotin-anti-IgE antibody for secondary antibody (manufactured byPharmingen) was added at 0.5 μg/ml and left at room temperature for 1hour. After washing with PBS-Tween, streptoavidin-HRP (manufactured byPharmingen) was added and left at room temperature for 30 minutes. Afterwashing with PBS-Tween, TMB+Substrate Chromogen (manufactured by DAKO)was added to initiate coloration reaction, After the colorationreaction, sulfuric acid (1 N) was added, and the absorbance (450 nm) wasmeasured with a microtiter plate, and the IgE level in serum wascalculated.

The change in dermatitis development score is shown in FIG. 1. The 5items: [1] dry skin and crust formation, [2] reddening and bleeding, [3]tissue dropping and scratch, [4] edema, and [5] itching behavior werescored in 4 ranks [0. no symptoms], [2. light], [3. moderate] and [4.severe] to calculate the total score in evaluation of each mouse. Thedevelopment score 13 days (Day 13) after induction of dermatitis hadbeen initiated was significantly lower in the OLS3059 administrationgroup than in the distilled water administration group. On the otherhand, the development score in the ATCC 53103 administration group wasnot significantly different from that in the distilled wateradministration group.

The total IgE level is shown in FIG. 2. The total IgE in serum 15 days(Day 15) after induction of dermatitis had been initiated wassignificantly lower in the OLS3059 administration group than in thedistilled water administration group. The total IgE in serum in the ATCC53103 administration group was slightly lower than that in the distilledwater administration group, but a significant difference therebetweenwas not recognized. ATCC 53103 is a lactic acid bacterium known to beeffective against atopic dermatitis, but OLS3059 had a higher inhibitoryeffect on development of atopic dermatitis than by ATCC 53103.

Example 2 Verification of Inhibitory Effect on Atopic Dermatitis in anIn Vivo Test

Four-week-old female NC/Nga mice (manufactured by Nippon SLC Co., Ltd.)were acclimated and raised and then divided into the following 3 groups:

a distilled water administration group (n=4),

a Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 (alsoreferred to hereinafter as OLL1073R-1) administration group (n=6),

and a Streptococcus thermophilus OLS3059 (isolated from Bulgarianyoghurt LB81 (registered trademark) manufactured by Meiji DairiesCorporation) administration group (n=5).

Feed (trade name: CRF-1, manufactured by Oriental Yeast Co., Ltd.) anddistilled water were freely given to the mice. The experimentalconditions were the same as in Example 1 except that the period ofinduction of dermatitis was 30 days.

The change in dermatitis development score is shown in FIG. 3. Thedevelopment score 13 days (Day 13) after induction of dermatitis hadbeen initiated was significantly lower in the OLL1073R-1 group and theOLS3059 administration group than in the distilled water administrationgroup.

The total IgE level is shown in FIG. 4. The total IgE in the OLL1073R-1group and in the OLS3059 administration group was significantly lowerthan in the distilled water administration group. The OLS3059 of thepresent invention was recognized to have an inhibitory effect ondevelopment of atopic dermatitis. The OLL1073R-1 of the presentinvention was also recognized to have an inhibitory effect ondevelopment of atopic dermatitis, but the IgE level was lower in theOLS3059 of the present invention.

Example 3 Verification of Inhibitory Effect on Atopic Dermatitis in anIn Vivo Test

The OLL1073R-1 of the present invention, which had been confirmed inExample 2 to exhibit an inhibitory effect comparable to that of theOLS3059 of the present invention on development of atopic dermatitis,was used in combination with the OLS3059 of the present invention, toexamine the inhibitory effect thereof on development of atopicdermatitis.

As a control for comparison with the combination of OLS3059 andOLL1073R-1 in the present invention, a combination of Lactobacillusdelbrueckii subspecies bulgaricus MEP1705601 and Streptococcusthermophilus MEP1705601, both of which were isolated from Bulgarianyoghurt (manufactured by Meiji Dairies Corporation), was used.

Four-week-old female NC/Nga mice (manufactured by Nippon SLC Co., Ltd.)were acclimated and raised and then divided into the following 4 groups.

That is, the mice were classified into a group given a fermented productproduced using Lactobacillus delbrueckii subspecies bulgaricusOLL1073R-1 and Streptococcus thermophilus OLS3059 (also referred tohereinafter as OLL1073R-1+OLS3059 administration group) (n=8), a groupgiven a fermented product produced using Lactobacillus delbrueckiisubspecies bulgaricus MEP1705601 and Streptococcus thermophilusMEP1705601 (also referred to hereinafter as LB MEP1705601+ST MEP1705601administration group) (n=9),

a non-fermented product administration group (n=9) and a dermatitisnon-induction, non-fermented product administration group (also referredto hereinafter as non-induction group) (n=4).

Either the fermented product or the non-fermented product was ingestedin an amount of 100 mg/mouse/day, and orally administered on consecutivedays until the day when the test was terminated.

The number of bacteria in the fermented product was 1×10⁹ cfu/g forOLL1073R-1, 3×10⁹ cfu/g for OLS3059, 8.6×10⁸ cfu/g for LB MEP1705601,and 2.1×10⁹ cfu/g for ST MEP1705601.

Each of the fermented product and the non-fermented product was preparedby lyophilization and suspended in distilled water beforeadministration. The experimental conditions were the same as in Example1 except that the dermatitis induction period was 29 days.

The change in dermatitis development score is shown in FIG. 5. Thedevelopment score 14 days (Day 14) after induction of dermatitis hadbeen initiated was significantly lower in the OLL1073R-1+OLS3059administration group than in the LB MEP1705601+ST MEP1705601administration group or in the non-fermented product administrationgroup.

The development score on days following 14 days (Day 14) after inductionof dermatitis had been initiated was significantly lower in theOLL1073R-1+OLS3059 administration group than in the LB MEP1705601+STMEP1705601 administration group or in the non-fermented productadministration group.

It was confirmed that OLL1073R-1, which had been confirmed in Example 2to exhibit an inhibitory effect comparable to that of the OLS3059 of thepresent invention on development of atopic dermatitis, can used incombination with the OLS3059 of the present invention, to exhibit aninhibitory effect on development of atopic dermatitis.

Example 4 Fermented Milk Production Example 1

50.0 g raw milk, 5.0 kg skim milk, 8.0 kg sugar, 1.0 kg collagen peptide(trade name: Nippi Peptide, manufactured by Nippi Collagen Cosmetics,Ltd.), 0.1 kg ceramide-containing konnyaku (mannan) potato extract(tradename: Konnyaku Ceramide, manufactured by Unitika Ltd.), 0.1 kgperfume, and 33.8 kg water were mixed and dissolved at a temperature of65° C. to prepare a milk preparation. This milk preparation wassubjected to homogenization treatment at a temperature of 65° C.(pressure 100 kg/cm²) and then sterilized at a temperature of 95° C. for2 minutes and immediately cooled to a temperature of 43 to 45° C. 2.0 kglactic acid bacterium starter was added to this sterilized milkpreparation which was then charged into a container, followed byfermentation at a temperature of 43° C. for 4 hours to give fermentedmilk (set yoghurt). The lactic acid bacterium starter used was a mixtureof equal volumes of Lactobacillus bulgaricus 2038 isolated fromBulgarian yoghurt LB81 (registered trademark) manufactured by MeijiDairies Corporation and Streptococcus thermophilus OLS3059 (alsoreferred to hereinafter as OLS3059, isolated from Bulgarian yoghurt LB81(registered trademark) manufactured by Meiji Dairies Corporation).

Example 5 Fermented Milk Drink Production Example 1

23.0 g raw milk, 12.1 kg skim milk and 62.9 kg water were mixed anddissolved at a temperature of 70° C. to prepare a milk preparation. Thismilk preparation was subjected to homogenization treatment at atemperature of 65° C. (pressure 150 kg/cm²) and then sterilized at atemperature of 95° C. for 10 minutes and immediately cooled to atemperature of 40 to 45° C. 2.0 kg lactic acid bacterium starter wasadded to this sterilized milk preparation, followed by fermentation at atemperature of 40 to 45° C. for 5 hours to give fermented milk.

The lactic acid bacterium starter used was a mixture of equal volumes ofLactobacillus bulgaricus 2038 isolated from Bulgarian yoghurt LB81(registered trademark) manufactured by Meiji Dairies Corporation andStreptococcus thermophilus OLS3059 (also referred to hereinafter asOLS3059, isolated from Bulgarian yoghurt LB81 (registered trademark)manufactured by Meiji Dairies Corporation).

For mechanically breaking curds of this fermented milk, the fermentedmilk was subjected to homogenization treatment (pressure 150 kg/cm²) andthen cooled to a temperature of 5° C. to give a fermented milkpreparation. Separately, 3.0 kg pectin, 2.0 kg sugar, 0.02 kg sweetener,0.1 kg perfume and 34.0 kg water were mixed and dissolved at atemperature of 65° C., then sterilized at a temperature of 130° C. for 2seconds, and immediately cooled to a temperature of 5° C. to form asugar solution.

The fermented milk preparation was mixed with the sugar solution at apredetermined concentration to give a fermented milk drink (drink-typeyoghurt).

Example 6 Fermented Milk Drink Production Example 2

23.0 g raw milk, 12.1 kg skim milk and 62.9 kg water were mixed anddissolved at a temperature of 70° C. to prepare a milk preparation. Thismilk preparation was subjected to homogenization treatment at atemperature of 65° C. (pressure 150 kg/cm²) and then sterilize datatemperature of 95° C. for 10 minutes and immediately cooled to atemperature of 40 to 45° C. 2.0 kg lactic acid bacterium starter wasadded to this sterilized milk preparation, followed by fermentation at atemperature of 40 to 45° C. for 5 hours to give fermented milk.

The lactic acid bacterium starter used was a mixture of equal volumes ofLactobacillus bulgaricus 2038 isolated from Bulgarian yoghurt LB81(registered trademark) manufactured by Meiji Dairies Corporation andStreptococcus thermophilus OLS3059 (also referred to hereinafter asOLS3059, isolated from Bulgarian yoghurt LB81 (registered trademark)manufactured by Meiji Dairies Corporation).

For mechanically breaking curds of this fermented milk, the fermentedmilk was subjected to homogenization treatment (pressure 150 kg/cm²) andthen cooled to a temperature of 5° C. to give a fermented milkpreparation. Separately, 3.0 kg pectin, 2.0 kg sugar, 0.02 kg sweetener,1.0 kg collagen peptide (trade name: Collagen Peptide 5200, manufacturedby Nitta Gelatin Co., Ltd.), 0.03 kg ceramide-containing corn extract(trade name: Corn Ceramide ME1-R, manufactured by Tsuji Seiyu Co.,Ltd.), 0.1 kg perfume and 34.0 kg water were mixed and dissolved at atemperature of 65° C., then sterilized at a temperature of 130° C. for 2seconds, and immediately cooled to a temperature of 5° C. to form asugar solution.

The fermented milk preparation was mixed with the sugar solution at apredetermined concentration to give a fermented milk drink (drink-typeyoghurt).

Example 7 Milk Drink Production Example

1 g lyophilized powder of Streptococcus thermophilus OLS3059 (alsoreferred to hereinafter as OLS3059, isolated from Bulgarian yoghurt LB81(registered trademark) manufactured by Meiji Dairies Corporation) wasadded to 1000 ml raw milk to prepare a milk preparation. This milkpreparation was subjected to homogenization treatment, then sterilizedat a temperature of 130° C. for 3 seconds and immediately cooled to atemperature of 10° C. or less. This sterilized milk preparation wascharged into a container to give a milk drink.

Example 8 Verification of Skin Improvement in a Human Test

The above fermented milk (Streptococcus thermophilus OLS3059; density ofbacteria, 85×10⁷ cfu/g; collagen peptide, 1000 mg/90 g; ceramide, 300μg/90 g) in Example 4 was used as a test meal in a human test where thetest meal was ingested twice (that is, morning and night respectively)per day for 28 days in an amount of 90 g for each ingestion. Thesubjects were 31 women (average age: 31.8 years old).

On the starting date (before ingestion of the test meal) and on the enddate (Day 28), the elasticity and texture density of the subjects' skinwere evaluated and observed, and in addition, the skin was observed foreruptions by a physician. The skin elasticity was determined byselecting one of right-and-left cheeks and measuring one site twice onthe cheek (trade name: CUTOMETER SEM575, manufactured byCourage+Khazaka). The texture density of the skin was determined byselecting a site of rough dry skin and measuring one site (trade name:Visual Imager VI-20, manufactured by Inforward, Inc.). Eruptions wereevaluated by determining the total number of eruptions.

On the starting date (before ingestion of the test meal) and on the enddate (Day 28), the subjects were surveyed by questionnaire. Thequestionnaire is shown in Table 1. As shown in Table 1, the 18 items:[1] skin gloss, [2] firmness, [3] dullness, [4] fleck, [5] sagging, [6]pores of the skin, [7] fine texture, [8] wrinkle (forehead), [9] wrinkle(around lips), [10] wrinkle (around eyes), [11] degree of dryness, [12]stickiness, [13] transparency, [14] redness, [15] spreading offoundation cream on the skin, [16] swelling, [17] constipation, and [18]dark circles under eyes, were evaluated in 5 ranks. On the end date, theusefulness of the test meal was evaluated by both the subject and aphysician.

TABLE 1 Items Point 5 Point 4 Point 3 Point 2 Point 1 1) skin glossglossy somewhat glossy moderately slight not glossy not glossy 2)firmness firm somewhat firm moderately slight not firm not firm 3)dullness not dull hardly dull moderately slight dull dull 4) fleck notflecked hardly flecked moderately slight flecked flecked 5) sagging notsagged hardly sagged moderately slight sagged sagged 6) pores of theskin unremarkable pores hardly remarkable moderately slight remarkableremarkable pores 7) fine texture fine somewhat fine moderately slightnot fine not fine 8) wrinkle not wrinkled hardly wrinkled moderatelyslight wrinkled wrinkled (forehead) 9) wrinkle (around not wrinkledhardly wrinkled moderately slight wrinkled wrinkled lips) 10) wrinkle(around not wrinkled hardly wrinkled moderately slight wrinkled wrinkledeyes) 11) degree of not dried hardly dried moderately slight dried welldried dryness 12) stickiness not sticky hardly sticky moderately slightsticky sticky 13) transparency transparent somewhat moderately slighttransparent not transparent transparent 14) redness not reddish hardlyreddish moderately slight reddish reddish 15) spreading of good slightlygood moderately slight bad bad foundation cream on the skin 16) swellingnot swollen hardly swollen moderately slight swollen well swollen 17)constipation not constipated hardly constipated moderately slightconstipated constipated 18) dark circles no dark circles scarce darkcircles moderate dark some dark circles considerable dark under eyescircles circles

The evaluation result of skin elasticity upon administration of the testmeal in Example 5 is shown in FIG. 6. By ingesting the test meal inExample 5, the skin elasticity was significantly increased, and the skinfirmness was improved. The evaluation result of texture density of theskin upon administration of the test meal in Example 5 is shown in FIG.7. By ingesting the test meal in Example 5, the texture density of theskin was significantly increased, and the texture density of the skinwas improved. The evaluation result of total number of eruptions uponadministration of the test meal in Example 5 is shown in FIG. 8. Byingesting the test meal in Example 5, the total number of eruptions wassignificantly decreased, and the rough surface was ameliorated.

The result of questionnaire survey of the subjects is shown in Table 2.It was confirmed that the skin condition and constipation of thesubjects are ameliorated by ingesting the test meal. The evaluationresult of usefulness of the test meal by both the subject and physicianis shown in FIG. 9. It was recognized by the subject or the physicianthat ingestion of the test meal is useful for the skin. The excellentskin improvement effect and/or treatment effect of Streptococcusthermophilus OLS3059 was demonstrated, and Streptococcus thermophilusOLS3059 was confirmed to be suitable for use in exhibiting the skinimprovement effect and/or treatment effect.

TABLE 2 Skin gloss Firmness Dullness Fleck Sagging Pores of the skinInitiation Initiation Initiation Initiation End Initiation EndInitiation End date End date date End date date End date date date datedate date date Point 5 (persons) 0 0 0 1 0 0 1 1 0 0 1 1 Point 4(persons) 1 13 2 13 0 7 1 3 1 7 0 4 Point 3 (persons) 8 14 6 10 3 14 0 53 10 3 7 Point 2 (persons) 14 4 16 7 18 8 14 11 19 11 12 14 Point 1(persons) 8 0 7 0 10 2 15 11 8 3 15 5 Total (persons) 31 31 31 31 31 3131 31 31 31 31 31 Average point 2.1 3.3 2.1 3.3 1.8 2.8 1.7 2.1 1.9 2.71.7 2.4 Wrinkle Wrinkle Wrinkle Degree of Fine texture (forehead)(around lips) (around eyes) dryness Stickiness Initiation InitiationInitiation Initiation End Initiation End Initiation End date End datedate End date date End date date date date date date date Point 5(persons) 0 0 1 0 0 1 0 1 0 0 3 1 Point 4 (persons) 3 10 4 12 3 8 3 8 415 3 11 Point 3 (persons) 8 12 7 10 7 9 6 5 6 4 3 7 Point 2 (persons) 127 13 7 16 11 11 12 10 12 12 10 Point 1 (persons) 8 2 6 2 5 2 11 5 11 010 2 Total (persons) 31 31 31 31 31 31 31 31 31 31 31 31 Average point2.2 3.0 2.4 3.0 2.3 2.8 2.0 2.6 2.1 3.1 2.3 3.0 Spreading of Darkcircles foundation cream under Transparency Redness on the skin SwellingConstipation eyes Initiation Initiation Initiation Initiation EndInitiation End Initiation End date End date date End date date End datedate date date date date date Point 5 (persons) 0 1 2 8 0 2 2 4 0 7 0 3Point 4 (persons) 1 5 9 12 1 19 5 13 1 13 3 7 Point 3 (persons) 6 11 8 410 7 5 4 0 3 3 4 Point 2 (persons) 14 12 8 7 13 3 11 9 19 5 11 10 Point1 (persons) 10 2 4 0 7 0 8 1 11 3 14 7 Total (persons) 31 31 31 31 31 3131 31 31 31 31 31 Average point 1.9 2.7 2.9 3.7 2.2 3.6 2.4 3.3 1.7 3.51.8 2.6

Example 9 Verification of the Relationship Between High IntestinalFunction Regulation Improvement and Skin Improvement Effect in a HumanTest

The fermented milk (Streptococcus thermophilus OLS3059; density ofbacteria, 50×10⁷ cfu/g) in Example 5 and the fermented milk(Streptococcus thermophilus OLS3059; density of bacteria, 50×10⁷ cfu/g;collagen peptide, 1000 mg/120 ml; ceramide, 300 μg/120 ml) in Example 6were used as test meals in a human test where the test meals wereingested twice (morning and night respectively) per day for 28 days inan amount of 120 g for each ingestion. The fermented milk drink inExample 5 was examined in 28 women as subjects (average age: 31.1 yearsold), and the fermented milk drink in Example 6 was examined in 28 womenas subjects (average age: 30.3 years old).

The questionnaire survey of the subjects was conducted in the samemanner as in Example 8. The questionnaire is as shown in Table 1. Theresults of questionnaire survey of the subjects are shown in Tables 3and 4. It was confirmed that the skin state and constipation of thesubjects are ameliorated by ingesting the test meals. The test meal(containing collagen and ceramide) in Example 6 had a particularlyhigher effect than by the test meal in Example 5 on 6 items in Table 1:that is, [1] skin gloss, [2] firmness, [7] fine texture, [11] degree ofdryness, [15] spreading of foundation cream on the skin, and [17]constipation.

TABLE 3 Collagen · peptide-“free” Skin gloss Firmness Dullness FleckSagging Pores of the skin Initiation Initiation Initiation InitiationEnd Initiation End Initiation End date End date date End date date Enddate date date date date date date Point 5 (persons) 0 0 0 0 0 0 0 2 0 01 0 Point 4 (persons) 2 9 3 9 2 7 6 6 3 7 3 4 Point 3 (persons) 7 10 1112 4 7 3 3 11 10 2 8 Point 2 (persons) 17 8 10 7 15 12 11 11 11 8 14 15Point 1 (persons) 2 1 4 0 7 2 8 6 3 3 8 1 Total (persons) 28 28 28 28 2828 28 28 28 28 28 28 Average point 2.3 3.0 2.5 3.1 2.0 2.7 2.3 2.5 2.52.8 2.1 2.5 Wrinkle Wrinkle Wrinkle Degree of Fine texture (forehead)(around lips) (around eyes) dryness Stickiness Initiation InitiationInitiation Initiation End Initiation End Initiation End date End datedate End date date End date date date date date date date Point 5(persons) 2 1 2 2 2 2 0 1 0 0 0 2 Point 4 (persons) 2 6 5 9 8 12 3 7 0 48 12 Point 3 (persons) 6 9 6 3 5 8 2 4 2 3 8 10 Point 2 (persons) 16 911 11 13 6 18 13 6 11 9 3 Point 1 (persons) 2 3 4 3 0 0 5 3 20 10 3 1Total (persons) 28 28 28 28 28 28 28 28 28 28 28 28 Average point 2.52.8 2.6 2.9 3.0 3.4 2.1 2.6 1.4 2.0 2.8 3.4 Spreading of Dark circlesfoundation cream under Transparency Redness on the skin SwellingConstipation eyes Initiation Initiation Initiation Initiation EndInitiation End Initiation End date End date date End date date End datedate date date date date date Point 5 (persons) 0 0 2 1 2 2 1 4 0 2 0 2Point 4 (persons) 2 4 7 7 2 11 6 10 0 9 2 6 Point 3 (persons) 3 13 2 5 58 3 6 0 4 6 3 Point 2 (persons) 20 10 13 14 17 7 14 6 10 10 5 7 Point 1(persons) 3 1 4 1 2 0 4 2 18 3 15 10 Total (persons) 28 28 28 28 28 2828 28 28 28 28 28 Average point 2.1 2.7 2.6 2.8 2.5 3.3 2.5 3.3 1.4 2.91.8 2.4

The change in intestinal function regulation (bowel movement) score isshown in Table 4. Scoring was conducted in the 5 ranks: “1. notconstipated”, “2. hardly constipated,” “3. moderate,” “4. slightlyconstipated,” and “5. constipated,” and the difference in each subjectbefore ingestion and after ingestion was calculated and evaluated. Forexample, when the subject whose had been “5. constipated” beforeingestion of the test meal became “3. moderate” after ingestion, theintestinal function regulation was analyzed as being improved by “2 ormore”.

TABLE 4 Collagen · peptide-“containing” Skin gloss Firmness DullnessFleck Sagging Pores of the skin Initiation Initiation InitiationInitiation End Initiation End Initiation End date End date date End datedate End date date date date date date date Point 5 (persons) 0 0 1 0 00 1 2 1 1 0 2 Point 4 (persons) 2 10 4 17 1 8 1 3 6 10 5 8 Point 3(persons) 9 14 8 7 1 11 2 7 5 7 6 3 Point 2 (persons) 11 2 9 3 21 8 1713 11 9 13 14 Point 1 (persons) 6 2 6 1 5 1 7 3 5 1 4 1 Total (persons)28 28 28 28 28 28 28 28 28 28 28 28 Average point 2.3 3.1 2.5 3.4 1.92.9 2.0 2.6 2.5 3.0 2.4 2.9 Wrinkle Wrinkle Wrinkle Degree of Finetexture (forehead) (around lips) (around eyes) dryness StickinessInitiation Initiation Initiation Initiation End Initiation EndInitiation End date End date date End date date End date date date datedate date date Point 5 (persons) 0 0 0 2 1 1 2 2 0 0 3 3 Point 4(persons) 5 9 10 9 8 15 6 12 0 13 11 15 Point 3 (persons) 8 14 4 5 5 3 35 0 2 6 5 Point 2 (persons) 11 4 9 9 10 8 12 8 10 10 8 5 Point 1(persons) 4 1 5 3 4 1 5 1 18 3 0 0 Total (persons) 28 28 28 28 28 28 2828 28 28 28 28 Average point 2.5 3.1 2.7 2.9 2.7 3.3 2.6 3.2 1.4 2.9 3.33.6 Spreading of Dark circles foundation cream under TransparencyRedness on the skin Swelling Constipation eyes Initiation InitiationInitiation Initiation End Initiation End Initiation End date End datedate End date date End date date date date date date date Point 5(persons) 0 1 2 2 0 2 0 3 0 7 1 1 Point 4 (persons) 2 5 11 10 5 16 11 150 5 3 6 Point 3 (persons) 8 15 4 3 6 8 1 3 1 5 2 6 Point 2 (persons) 146 7 11 15 2 15 5 17 8 12 13 Point 1 (persons) 4 1 4 2 2 0 1 2 10 3 10 2Total (persons) 28 28 28 28 28 28 28 28 28 28 28 28 Average point 2.33.0 3.0 3.0 2.5 3.6 2.8 3.4 1.7 3.2 2.0 2.7

The results of evaluation of skin elasticity upon ingestion of the testmeals in Examples 5 and 6 are shown in FIGS. 10 to 12. FIG. 10 shows theresult of evaluation of the skin elasticity of the subject havingintestinal function regulation improved by “1 or more”, FIG. 11 showsthe result of evaluation of the skin elasticity of the subject havingintestinal function regulation improved by “2 or more”, and FIG. 12shows the result of evaluation of the skin elasticity of the subjecthaving intestinal function regulation improved by “3 or more”. It wasdemonstrated that for the human for whom the test meal exhibited ahigher intestinal function regulation (bowel movement) improvementeffect, the skin improvement of the test meal was higher. This tendencywas made more significant by the test meal (containing collagen peptideand ceramide) in Example 6 than by the test meal in Example 5. Byingesting the test meals in Examples 5 and 6, the elasticity of the skinwas increased, and the firmness of the skin was improved.

The results of evaluation of the texture density of skin upon ingestionof the test meals in Examples 5 and 6 are shown in FIGS. 13 to 15. FIG.13 shows the result of evaluation of the texture density of the skin ofthe subject having intestinal function regulation improved by “1 ormore”, FIG. 14 shows the result of evaluation of the texture density ofthe skin of the subject having intestinal function regulation improvedby “2 or more”, and FIG. 15 shows the result of evaluation of thetexture density of the skin of the subject having intestinal functionregulation improved by “3 or more”. It was demonstrated that for thehuman for whom the test meal exhibited a higher intestinal functionregulation (bowel movement) improvement effect, the skin improvement ofthe test meal was higher. This tendency was made more significant by thetest meal (containing collagen peptide and ceramide) in Example 4 thanby the test meal in Example 5. By ingesting the test means in Examples 5and 6, the texture density of the skin was increased, and the finetexture was improved.

INDUSTRIAL APPLICABILITY

According to the present invention, there can be provided a compositionand fermented milk which are derived from natural products, are highlysafe and have a high skin improvement effect and/or treatment effect,comprising lactic acid bacteria excellent in manufacturability.According to the present invention, there can be provided fermented milkhaving a higher skin improvement effect and treatment effect on humansfor whom it exhibited a higher intestinal function regulationimprovement effect and/or treatment effect.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing a change in dermatitis development score(expressed in average±standard deviation; **P<0.01, *P<0.05, Dunnett'smultiple comparison) during a test period in a distilled wateradministration group, a Lactobacillus rhamnosus ATCC 53103administration group, a Streptococcus thermophilus OLS3059administration group and a dermatitis non-induction group.

FIG. 2 is a graph showing total IgE level in serum (expressed inaverage±standard deviation; **P<0.01, Dunnett's multiple comparison)after 15 days (on Day 15) in a distilled water administration group, aLactobacillus rhamnosus ATCC 53103 administration group, a Streptococcusthermophilus OLS3059 administration group and a dermatitis non-inductiongroup.

FIG. 3 is a graph showing a change in dermatitis development score(expressed in average±standard deviation; **P<0.01, *P<0.05, Dunnett'smultiple comparison) during a test period in a distilled wateradministration group, a Lactobacillus bulgaricus OLL1073R-1administration group and a Streptococcus thermophilus OLS3059administration group.

FIG. 4 is a graph showing total IgE level in serum (expressed inaverage±standard deviation; **P<0.01, Dunnett's multiple comparison)after 15 days (on Day 15) in a distilled water administration group, aLactobacillus bulgaricus OLL1073R-1 administration group and aStreptococcus thermophilus OLS3059 administration group.

FIG. 5 is a graph showing a change in dermatitis development score(expressed in average±standard deviation; **P<0.01, *P<0.05, Dunnett'smultiple comparison) during a test period in a group administered with afermented product produced with Lactobacillus bulgaricus OLL1073R-1 andStreptococcus thermophilus OLS3059, a group administered with afermented product produced with Lactobacillus delbrueckii subspeciesbulgaricus MEP1705601 and Streptococcus thermophilus MEP1705601.

FIG. 6 is a graph showing the elasticity of the skin of a subject whoingested, as test meal, fermented milk produced with Streptococcusthermophilus OLS3059.

FIG. 7 is a graph showing the texture density of the skin of a subjectwho ingested, as test meal, fermented milk produced with Streptococcusthermophilus OLS3059.

FIG. 8 is a graph showing the observation result of the total number oferuptions in a subject who ingested, as test meal, fermented milkproduced with Streptococcus thermophilus OLS3059.

FIG. 9 is a graph showing the evaluation result of usefulness by aphysician and by a subject who ingested, as test meal, fermented milkproduced with Streptococcus thermophilus OLS3059.

FIG. 10 is a graph showing the elasticity of the skin of a subject with“intestinal function regulation improved by ‘1 or more’”, who ingested,as test meal, fermented milk produced with Streptococcus thermophilusOLS3059.

FIG. 11 is a graph showing the elasticity of the skin of a subject with“intestinal function regulation improved by ‘2 or more’”, who ingested,as test meal, fermented milk produced with Streptococcus thermophilusOLS3059.

FIG. 12 is a graph showing the elasticity of the skin of a subject with“intestinal function regulation improved by ‘3 or more’”, who ingested,as test meal, fermented milk produced with Streptococcus thermophilusOLS3059.

FIG. 13 is a graph showing the texture density of the skin of a subjectwith “intestinal function regulation improved by ‘1 or more’”, whoingested, as test meal, fermented milk produced with Streptococcusthermophilus OLS3059.

FIG. 14 is a graph showing the texture density of the skin of a subjectwith “intestinal function regulation improved by ‘2 or more’”, whoingested, as test meal, fermented milk produced with Streptococcusthermophilus OLS3059.

FIG. 15 is a graph showing the texture density of the skin of a subjectwith “intestinal function regulation improved by ‘3 or more’”, whoingested, as test meal, fermented milk produced with Streptococcusthermophilus OLS3059.

1. Use of Streptococcus thermophilus OLS3059 in skin improvement and/ortreatment.
 2. A composition for skin improvement and/or treatment, whichcomprises Streptococcus thermophilus OLS3059 and/or a culture thereof.3. The composition for skin improvement and/or treatment according toclaim 2, which further comprises Lactobacillus delbrueckii subspeciesbulgaricus OLL1073R-1 and/or a culture thereof.
 4. Food and drinkcomprising the composition of claim
 2. 5. Fermented milk for skinimprovement and/or treatment prepared by using Lactobacillus delbrueckiisubspecies bulgaricus and Streptococcus thermophilus OLS3059.
 6. Thefermented milk for skin improvement and/or treatment according to claim5, which further comprises collagen peptide and/or ceramide.
 7. Aprocess for producing the fermented milk for skin improvement and/ortreatment of claim 5, which comprises preparing raw milk, addingLactobacillus delbrueckii subspecies bulgaricus and Streptococcusthermophilus OLS3059 as starters to the milk, charging the resultingmilk into a container, and fermenting it to form curd followed bycold-storage thereof.
 8. A process for producing the fermented milk forskin improvement and/or treatment of claim 5, which comprises preparingraw milk, adding Lactobacillus delbrueckii subspecies bulgaricus andStreptococcus thermophilus OLS3059 as starters to the milk, fermentingthe resulting milk to form curd, disrupting the curd, and charging itinto a container followed by cold-storage thereof.
 9. A process forproducing the fermented milk for skin improvement and/or treatment ofclaim 5, which comprises preparing raw milk, adding Lactobacillusdelbrueckii subspecies bulgaricus and Streptococcus thermophilus OLS3059as starters to the milk, fermenting the milk to form curd, disruptingthe curd, cooling it, and charging it into a container followed bycold-storage thereof.
 10. Food and drink comprising the composition ofclaim
 3. 11. A process for producing the fermented milk for skinimprovement and/or treatment of claim 6, which comprises preparing rawmilk, adding Lactobacillus delbrueckii subspecies bulgaricus andStreptococcus thermophilus OLS3059 as starters to the milk, charging theresulting milk into a container, and fermenting it to form curd followedby cold-storage thereof.
 12. A process for producing the fermented milkfor skin improvement and/or treatment of claim 6, which comprisespreparing raw milk, adding Lactobacillus delbrueckii subspeciesbulgaricus and Streptococcus thermophilus OLS3059 as starters to themilk, fermenting the resulting milk to form curd, disrupting the curd,and charging it into a container followed by cold-storage thereof.
 13. Aprocess for producing the fermented milk for skin improvement and/ortreatment of claim 6, which comprises preparing raw milk, addingLactobacillus delbrueckii subspecies bulgaricus and Streptococcusthermophilus OLS3059 as starters to the milk, fermenting the milk toform curd, disrupting the curd, cooling it, and charging it into acontainer followed by cold-storage thereof.